Antigenic and genomic characterization of adenovirus associated to respiratory infections in children living in Northeast Brazil

From January to December 1998, nasopharyngeal aspirates were obtained from 482 children with acute respiratory infections attended in emergence department and wards of a teaching hospital in the city of Salvador, Brazil. The samples were tested for the presence of adenovirus by isolation in tissue culture and indirect immunofluorescence assay. Eleven adenoviruses were detected by both methods in the same clinical samples. Infections by adenovirus were observed during seven months of the year without association with rainy season. Genome analysis was performed on these 11 isolates. Species C was represented by serotypes 1, 2 and 5. Within species B, only serotype 7 (Ad7) was detected. Two genomic variants of Ad1, two variants of Ad2, one of Ad5, and one of Ad7 (7h) were identified. This is the first study of molecular epidemiology of adenovirus associated to acute respiratory infections in children living in Northeast Brazil, and contributes to a better understanding of adenovirus infections in the country.

ANATOMICAL STUDIES OF IN VITRO ORGANOGENESIS INDUCED IN LEAF-DERIVED EXPLANTS OF PASSIONFRUIT

With the aim of studying the organogenesis in vitro in Passiflora edulis Sims f. flavicarpa Deg., the passionfruit, leaf-derived explants were cultured on media containing NAA or BAP and incubated either in continuous darkness or in light. The histological events leading to de novo organ formation were evaluated. Darkness induces rhizogenesis in the presence of NAA, whereas direct shoot regeneration is stimulated by light and BAP. This latter condition is recommended for passionfruit micropropagation as several adventitious shoot buds were formed from meristemoids of parenchymal origin.

Development of blast resistant somaclones of the upland rice cultivar araguaia

The degree of blast resistance of upland rice (Oryza sativa L.) cultivar Araguaia has decreased over time causing significant yield losses. The major objective of this study was to obtain blast (Pyricularia grisea) resistant somaclones, adapting greenhouse and field selection procedures. Rice blast resistance and agronomic traits were assessed in R2 to R6 generations derived from regenerant plants (R1) from immature panicles of Araguaia. The evaluation and selection procedures include testing of early segregating populations and fixed lines in the advanced generations, under natural field conditions, and artificial inoculations in the greenhouse, with prevalent races IB-1 and IB-9 of P. grisea. Somaclones with both vertical resistance and slow blasting resistance were obtained. Twenty of 31 somaclones developed with a high degree of vertical resistance and fan shaped plant type maintained resistance in field and blast nursery tests in the R6 generation. Greenhouse selection with two specific physiologic races yielded 44 somaclones with slow blasting resistance, similar plant type and yield potential as that of Araguaia.

Plant regeneration from protoplast of Brazilian citrus cultivars

A procedure is described to regenerate plants from protoplasts of Brazilian citrus cultivars, after isolation, fusion and culture. Protoplasts were isolated from embryogenic cell suspension cultures and from leaf mesophyll of seedlings germinated in vitro. The enzyme solution for protoplast isolation was composed of mannitol (0.7 M), CaCl2 (24.5 mM), NaH2PO4 (0.92 mM), MES (6.15 mM), cellulase (Onozuka RS - Yakult, 1%), macerase (Onozuka R10 - Yakult, 1%) and pectolyase Y-23 (Seishin, 0.2%). Protoplast culture in liquid medium after chemical fusion lead to the formation of callus colonies further adapted to solid medium. Somatic embryo formation occurred spontaneously after two subcultures, on modified MT medium supplemented with 500 mg/L of malt extract. Well defined embryos were germinated in modified MT medium with addition of GA3 (2.0 muM) and malt extract (500 mg/L). Plant regeneration was also achieved by adventitious shoots obtained through direct organogenesis of not well defined embryos in modified MT medium with addition of malt extract (500 mg/L), BAP (1.32 muM), NAA (1.07 muM) and coconut water (10 mL/L). Plantlets were transferred to root medium. Rooted plants were transferred to a greenhouse for further adaptation and development.

Sucrose metabolizing enzymes in cell suspension cultures of Bauhinia forficata, Curcuma zedoaria and Phaseolus vulgaris

The objective of this work was to study the activity of sucrose metabolizing enzymes in extracts of cell suspension cultures of Bauhinia forficata Link, Curcuma zedoaria Roscoe and Phaseolus vulgaris L. Invertase pathway was identified in the three studied species. Sucrose synthase pathway was also responsible for sucrose metabolism in Curcuma zedoaria and Phaseolus vulgaris cells. Activity values higher than 300 nmol min-1 mg-1 of protein were found for acid and neutral invertases, UDPglucose pyrophosphorylase and phosphoglucomutase in the cell extract of the three plant species. Sucrose synthase showed low activity in Bauhinia forficata cells. As sucrose concentration in the culture medium decreased, sucrose synthase activity increased in C. zedoaria and P. vulgaris cells. The glycolytic enzymes activity gradually reduced at the end of the culture period, when carbohydrate was limited.

Macropropagation and micropropagation of Ziziphus spina-christi

Christ's thorn (Ziziphus spina-christi (L.) Desf.) is a cross-pollinated plant with a wide range of genetic variability in nature and, for this reason, vegetative propagation assumes importance for improvement programs. The objective of this work was to evaluate cutting, T budding and tissue culture methods for this species. Shoots of 22-25 cm length were treated by two culture media and three shoot diameters for cutting trial. The T budding treatments consisted of three and five collection dates in spring and autumn, respectively. Tissue culture nodal segments bearing axillary buds were removed from shoots of mature trees at different seasons. Experiments to determine the best disinfectant chemical, appropriate conditions and materials to prevent phenolic compound exudation, explant characteristics, media type and cytokinin-auxin ratios were carried out. Successful rooting happened only on the sand beds and with cuttings greater than 8 mm diameter. The effects of T budding seasons on budtake percentage were significantly different. The best time for explant harvesting was mid of summer. Amount of rooting on media containing IBA as well as activated charcoal and disinfection with Ca(OCl)2 at concentration of 5% for 20 minutes were the best treatments.

Optimization of a protocol for the micropropagation of pineapple

The present work aimed at maximizing the number of plantlets obtained by the micropropagation of pineapple (Ananas comosus (L.) Merrill) cv. Pérola. Changes in benzylaminopurine (BAP) concentration, type of medium (liquid or solidified) and the type of explant in the proliferation phase were evaluated. Slips were used as the explant source, which consisted of axillary buds obtained after careful excision of the leaves. A Sterilization was done in the hood with ethanol (70%), for three minutes, followed by calcium hypochlorite (2%), for fifteen minutes, and three washes in sterile water. The explants were introduced in MS medium supplemented with 2mg L-1 BAP and maintained in a growth room at a 16h photoperiod (40 mmol.m-2.s-1), 27 ± 2ºC. After eight weeks, cultures were subcultured for multiplication in MS medium. The following treatments were tested: liquid x solidified medium with different BAP concentrations (0.0, 1.5 or 3.0 mg L-1), and the longitudinal cut, or not, of the shoot bud used as explant. The results showed that liquid medium supplemented with BAP at 1.5 mg L-1, associated with the longitudinal sectioning of the shoot bud used as explant presented the best results, maximizing shoot proliferation. On average, the best treatment would allow for an estimated production of 161,080 plantlets by the micropropagation of the axillary buds of one plant with eight slips and ten buds/slips, within a period of eight months.

Somatic embryogenesis from zygotic embryos of Euterpe oleracea Mart.

The aim of this work was to study the morphogenetic responses of zygotic embryos of açai palm (Euterpe oleracea Mart.) submitted to several conditions of in vitro culture. Several research experiments were conducted, in laboratory, using vegetable material collected from açai palm plants at Embrapa Amazon Oriental, Belém-PA, Brazil. It was possible to verify the expression of a direct, repetitive and no-synchronized model of somatic embryogenesis in mature zygotic embryos cultivated in primary MS medium supplemented with 2,4-D (339.36 muM) and transferred to a secondary MS medium in the presence of NAA (0.537 muM) and 2iP (12.30 muM). The conversion of somatic embryos into seedlings was reached after 210 days with the transfer of the cultures to a third medium with sucrose and mineral salts concentrations reduced to a half, without growth regulators.

Development of globular embryos from the hybridization between 'Pêra Rio' sweet orange and 'Poncã' mandarin

This research was undertaken to study the influence of different concentrations of the MT medium, sucrose, vitamins, activated charcoal and gibberellic acid (GA3) on the culture of immature embryos from the crossing between 'Pêra Rio' sweet orange and 'PONCÃ' mandarin. The embryos were excised under aseptic conditions and inoculated in 15 mL of the MT medium according to the following experiments: 1) MT concentrations (0%, 50%, 100%, 150% and 200%) supplemented with 0, 30, 60 and 90 g.L-1 of sucrose; 2) vitamins concentrations of the MT (0%, 50%, 100%, 150% and 200%) supplemented with 0, 30, 60 and 90 g.L-1 of sucrose; 3) activated charcoal concentrations (0, 0.5, 1, 1.5 and 2 g.L-1) supplemented with GA3 (0, 0.01, 0.1; 1 and 10 mg.L-1). After the inoculation, the embryos were kept in a growth room for 90 days at 27 ± 1ºC, in a 16-hour photoperiod with 32 µmol.m-2.s-1 of irradiance. The best development of embryos at the globular stage was achieved using 50% and 100% of the MT medium plus 60 g.L-1 and 90 g.L-1 of sucrose, respectively, supplemented with 0.01 mg.L-1 of GA3. The addition of activated charcoal or vitamins in the MT medium has shown to be unnecessary to the development of globular embryos.

Induction of somatic embryogenesis in immature seeds of guavatree cv. Paluma

The biotechnological techniques may help solve many problems of guava culture, such as the high perishability of fruits. Somatic embryogenesis can generate highly multiplicative cell cultures and with high regenerative potential, serving as basis for genetic transformation. The aim of this work was to obtain somatic embryogenesis of guava (Psidium guajava L.) cv. Paluma. Immature seeds were used, and they were inoculated in MS environment containing 400 mg L-1 of L-glutamine, 100 mg L-1 myo-inositol, 60 g L-1 sucrose, 100 mg L-1 ascorbic acid and supplemented with different types and concentrations of growth regulators. Embryogenic callus appeared after 37 days of culture in an environment containing 1.0 mg L-1 2.4-D + 2.0 mg L-1 2-ip, in 7% of the explants. After 65 days of culture, the treatment containing 0.5 mg L-1 CPA showed 20% of explants with direct embryos, while the treatment with 1 mg L-1 had 14% of explants with direct embryos and 7% of explants with embryogenic callus. In 66.6% of embryos regenerated with 0.5 mg L¹ CPA there was the formation of secondary embryos. The use of IASP and BAP, aiming embryogenesis proliferation, led to an increase in the cellular proliferation, but calli apparently lost their embryogenic potential.

In vitro organogenesis of rangpur lime

Rangpur lime (Citrus limonia Osbeck) in vitro organogenesis was studied based on explant type and cytokinin culture media supplementation. Four explants types collected from epicotyl or hypocotyl regions of in vitro germinated seedlings were evaluated. The epicotyl-derived explants consisted of epicotyl segments and the hypocotyl-derived explants consisted of the entire hypocotyl segment, the hypocotyl segment attached to a cotyledon fragment, and the hypocotyl segment divided longitudinally. The explants were cultured on EME culture medium supplemented with benzylaminopurine (0, 0.5, 1.0, or 1.5 mg L-1). The evaluation was performed after 6 weeks. Best results considering both the explant responsiveness and number of shoots developed per explants were obtained when epicotyl segments-derived explants were evaluated. Considering the explant responsiveness of hypocotyl segments-derived explants no difference was detected between the entire hypocotyl segment and the hypocotyl segment attached to a cotyledon fragment. Moreover, the percentage of responsive explants decreased when hypocotyl segments divided longitudinally were tested. No difference was detected for the number of shoots developed per explant considering the three types of hypocotyl-derived explants. Culture media supplementation with BAP was not essential for Rangpur lime in vitro organogenesis. However, adventitious shoot development was stimulated in concentrations between 0.5 - 1.0 mg L-1.

Production and quality of Anacardium othonianum Rizz. seedlings grown in different substrates

This study aimed to evaluate the effect of substrate on growth, emergence, nutrition, and quality of Anacardium othonianum Rizz. (cerrado cashew tree) seedlings. The experiment was conducted in a greenhouse at the Plant Tissue Culture Laboratory on the Rio Verde campus. The following substrates were used: 1) Bioplant®, 2) Mecplant® (MP) + carbonized rice husk (CRH) (7:3), 3) fine-grained vermiculite (FGV), 4) FGV+CRH (3:1), 5) FGV+CRH (1:1), 6) FGV+CRH (1:3), and 7) sugarcane bagasse (SB) + sugarcane mill filter cake (FC) (3:2). Emerged seedlings were counted at 2-day intervals for 38 days following emergence of the first seedling. At 39, 64, and 89 days after seeding (DAS), the following variables were measured: stem length (SL), stem diameter (SD), and number of leaves (NL). Accumulated dry weight, quality indices, and leaf macro- and micronutrient levels were determined at 89 DAS. Plants grown in the FGV and FGV+CFH (1:3) substrates had shorter stem lengths than the plants grown in other substrates. Increases in seedling growth were smaller between 64 and 89 DAS compared to the initial period of the experiment. The highest leaf N concentrations were found in the SB+FC substrate treatment group; P and K concentrations were higher for the MP+CRH (7:3), SB+FC, and Bioplant® treatments; and Ca levels were higher for the SB+FC and MP+CRH (7:3) substrate treatments. The MP+CRH (7:3) substrate treatment group had the highest leaf B and Mn micronutrient concentrations, and plants from the Bioplant® substrate group had the highest leaf B micronutrient content. Mg, S, Cu, Zn, and Fe concentrations did not differ among the different substrates. The plant traits that differed most among the treatments included stem length for the FGV and FGV+CRH (1:3) substrate groups and leaf nutrient concentrations, which were higher for the SB+FC group followed by the MP+CRH and Bioplant® treatments.

Biotechnology applied to Annona species: a review

Annonaceae is an ancient family of plants including approximately 50 genera growing worldwide in a quite restricted area with specific agroclimatic requirements. Only few species of this family has been cultivated and exploited commercially and most of them belonging to the genus Annona such as A. muricata, A. squamosa, the hybrid A. cherimola x A. squamosa and specially Annona cherimola: the cherimoya, commercially cultivated in Spain, Chile, California, Florida, México, Australia, Ecuador, Peru, Brazil, New Zealand and several countries in South and Central America. The cherimoya shows a high degree of heterozygosis, and to obtain homogeneous and productive orchards it is necessary to avoid the propagation by seeds of this species. Additionally, the traditional methods of vegetative propagation were inefficient and inadequate, due to the low morphogenetic potential of this species, and the low rooting rate. The in vitro tissue culture methods of micropropagation can be applied successfully to cherimoya and other Annona sp to overcome these problems. Most of the protocols of micropropagation and regeneration were developed using the cultivar Fino de Jete, which is the major cultivar in Spain. First it is developed the method to micropropagate the juvenile material of cherimoya (ENCINA et al., 1994), and later it was optimized a protocol to micropropagate adult cherimoya genotypes selected by outstanding agronomical traits (PADILLA and ENCINA, 2004) and further it was improved the process through micrografting (PADILLA and ENCINA, 2011).At the present time we are involved in inducing and obtaining new elite genotypes, as part of a breeding program for the cherimoya and other Annonas, using and optimizing different methodologies in vitro: a) Adventitious organogenesis and regeneration from cellular cultures (ENCINA, 2004), b) Ploidy manipulation of the cherimoya, to obtain haploid, tetraploid and triploid plants (seedless), c) Genetic transformation, for the genes introduction to control the postharvest processes and the genes introduction to provide resistance to pathogen and insects and d) Micropropagation and regeneration of other wild Annona or related Annonaceae species such as: Annona senegalensis, A. scleroderma, A. montana, A. reticulata, A. glabra, A. diversifolia and Rollinia sp.

Técnica de obtenção de filtrado de cultura de Alternaria solani

This work was developed to adapt culture filtrates of Alternaria solani to be used in vitro selection of resistant potato. Three isolates of A. solani (I1 and I2) from Eldorado do Sul and Rio Pardo were used. Two liquid media, V8 and Czapek, were used to grow each of the fungal isolate, giving six culture filtrates (I1V8, I2V8, I3V8, I1Cz, I2Cz and I3Cz). Two sterilization forms, Millipore and autoclave were tested. There was no difference in these two sterilization forms. Tissue culture and toxic filtrates of A. solani have a potential to reduce the time in selection of resistant potato.

In vitro multiplication of Tabernaemontana fuchsiaefolia L. (Apocynaceae)

This study describes a simple and promising for in vitro multiplication of Tabernaemontana fuchsiaefolia, a species abundantly found in southern Brazil utilized for medicinal purposes and as a source of compounds that may be used to develop new synthetic drugs. Apical and hypocotyl explants were cultured in MS medium containing different concentrations of the cytokinins benzylaminopurine (BA) and 6-furfurylaminopurine (kinetin), supplemented with phloroglucinol (1, 3, 5-hydroxybenzene) to stimulate growth and shoot proliferation. Cytokinin added to the culture media positively influenced the micropropagation of T. fuchsiaefolia.and kinetin induced more shoots per explant than BA cytokinin. A favorable effect of phloroglucinol on apical and lateral buds from hypocotyls was also achieved in medium containing no kinetin or in all kinetin concentrations tested. Short pulses of auxin 3-indolebutyric acid (IBA) 5.0 mg/l resulted in satisfactory rooting in apical microcuttings. The addition of phloroglucinol to MS medium induced rhizogenesis in 29% of the nodal segments transferred to MS medium in the absence of IBA and in 50% of the nodal segments transferred to MS medium containing 0.5 mg/l IBA and in nodal segments previously submitted to short pulses of IBA.